In many fields of life sciences research, including biological, biomedical, genetic, fermentation, aquaculture, agricultural, forensic and environmental research, there is a need to identify nucleic acids, qualitatively and quantitatively, in pure solutions and in biological samples. Such applications require a fast, sensitive, and selective methodology that can detect minute amounts of nucleic acids in a variety of media, whether or not the nucleic acid is contained in cells.
Although certain unsymmetrical cyanine dyes were first described before the genetic role of nucleic acids was established (Brooker, et at., J. AM. CHEM. SOC. 64, 199 (1942)), some unsymmetrical cyanine dyes are now known as effective fluorescent stains of DNA and RNA. The compound sold as Thiazole Orange has particular advantages in reticulocyte analysis (U.S. Pat. No. 4,883,867 to Lee, et at. (1989)) or in preferentially staining bloodborne parasites (U.S. Pat. No. 4,937,198 to Lee, et al. (1990)). Thiazole Orange readily stains many mammalian cells, yet does not effectively stain some eukaryotic cells.
Attachment of various cyclic structures to the pyrdinium or quinolinium ring system of the unsymmetrical cyanine dye was found to make the nucleic acid stains highly permeant to gels and a wider variety of cell types, including both gram-positive and gram-negative bacteria, yeasts, and eukaryotic cells as well as prokaryotic cells, as described in copending applications CYCLIC-SUBSTITUTED UNSYMMETRICAL CYANINE DYES (Ser. No. 08/090,890 to Haugland, et al. filed Jul. 12, 1993), FLUORESCENT ASSAY FOR BACTERIAL GRAM REACTION (Ser. No. 08/146,328 to Roth et at. filed Nov. 1, 1993), FLUORESCENT VIABILITY ASSAY USING CYCLIC-SUBSTITUTED UNSYMMETRICAL CYANINE DYES (Ser. No. 08/148,847 to Millard, et al. filed Nov. 8, 1993), and VIABILITY ASSAY FOR YEAST AND OTHER FUNGI (Ser. No. 08/206,081 to Roth, et al. filed Mar. 3, 1994); and PCT application 94/04127 (the specification of which is incorporated by reference).
Attachment of a cationic side chain at the nitrogen of the pyridinium or quinolinium ring system of the unsymmetrical cyanine dyes, on the other hand, was shown to make the stains relatively impermeant to all cells, except cells, particularly mammalian cells, where cell membrane integrity was destroyed, as described in UNSYMMETRICAL CYANINE DYES WITH CATIONIC SIDE CHAINS (U.S. Pat. No. 5,321,130 to Yue et al. (1994)). A second type of dye, in which a dye monomer is attached at the nitrogen of the quinolinium or pyridinium ring system to form dimeric compounds as described in DIMERS OF UNSYMMETRICAL CYANINE DYES (PCT 92/07867) and DIMERS OF UNSYMMETRICAL CYANINE DYES CONTAINING PYRIDINIUM MOlETIES (Ser. No. 08/043,665 to Yue, et al. filed Apr. 5, 1993) that are also relatively impermeant to all cells unless the cell membrane has been disrupted. Although these impermeant dyes were found to have the further advantage of increased binding affinity for nucleic acids, resulting in increased sensitivity for detection of cell free nucleic acids, a number of these dyes were also found to have a number of disadvantages for some applications, including a slow rate of equilibrium binding, electrostatic attraction to glass surfaces, moderate salt sensitivity, reduced photostability, lower quantum yield, relatively lower sensitivity of detection of nucleic acids in gelse and in solutions, and limited permeability to dead prokaryotic cells.
The dyes of the present invention are unsymmetrical cyanine dyes containing a defined substituent on the pyridinium or quinolinium ring system or a substituent immediately adjacent to the nitrogen atom of the pyridinium or quinolinium ring that modifies the permeability, selectivity and affinity of the dye for nucleic acids. Members of this class of dyes are more effective in detection of cell membrane integrity and in the staining or detection of nucleic acids, including DNA and RNA, in gels and in solutions, and in living and dead cells. Dyes substituted at the position adjacent to the ring nitrogen generally have unexpectedly higher quantum yields than dyes not substituted at that position. In addition, the ring substituent is easily modified, particularly by inclusion of an appropriate heteroatom in the substituent, to allow selectable alteration of the permeability and affinity of the dyes. Furthermore, by simple synthetic modification, a family of dyes having absorption and emission spectral properties that cover most of the visible and near-infrared spectrum can be prepared. Selection of an appropriately substituted dye enhances the sensitivity of analysis of nucleic acids utilizing a variety of techniques.